Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.     

Preclinical and clinical development of plant-made virus-like particle vaccine against avian H5N1 influenza

The recent swine H1N1 influenza outbreak demonstrated that egg-based vaccine manufacturing has an Achille's heel: its inability to provide a large number of doses quickly. Using a novel manufacturing platform based on transient expression of influenza surface glycoproteins in Nicotiana benthamiana, we have recently demonstrated that a candidate Virus-Like Particle (VLP) vaccine can be generated within 3 weeks of release of sequence information. Herein we report that alum-adjuvanted plant-made VLPs containing the hemagglutinin (HA) protein of H5N1 influenza (A/Indonesia/5/05) can induce cross-reactive antibodies in ferrets. Even low doses of this vaccine prevented pathology and reduced viral loads following heterotypic lethal challenge. We further report on safety and immunogenicity from a Phase I clinical study of the plant-made H5 VLP vaccine in healthy adults 18-60 years of age who received 2 doses 21 days apart of 5, 10 or 20 μg of alum-adjuvanted H5 VLP vaccine or placebo (alum). The vaccine was well tolerated at all doses. Adverse events (AE) were mild-to-moderate and self-limited. Pain at the injection site was the most frequent AE, reported in 70% of vaccinated subjects versus 50% of the placebo recipients. No allergic reactions were reported and the plant-made vaccine did not significantly increase the level of naturally occurring serum antibodies to plant-specific sugar moieties. The immunogenicity of the H5 VLP vaccine was evaluated by Hemagglutination-Inhibition (HI), Single Radial Hemolysis (SRH) and MicroNeutralisation (MN). Results from these three assays were highly correlated and showed similar trends across doses. There was a clear dose-response in all measures of immunogenicity and almost 96% of those in the higher dose groups (2 × 10 or 20 μg) mounted detectable MN responses. Evidence of striking cross-protection in ferrets combined with a good safety profile and promising immunogenicity in humans suggest that plant-based VLP vaccines should be further evaluated for use in pre-pandemic or pandemic situations. TRIAL REGISTRATION: ClinicalTrials.gov NCT00984945.     

Nelfinavir,an HIV-1 Protease Inhibitor,Induces Oxidative Stress–Mediated,Caspase-Independent Apoptosis in Leishmania Amastigotes

Background

Visceral leishmaniasis has now emerged as an important opportunistic disease in patients coinfected with human immunodeficiency virus type-1 (HIV-1). Although the effectiveness of HIV-1 protease inhibitors, such as nelfinavir, in antiretroviral therapies is well documented, little is known of the impact of these drugs on Leishmania in coinfected individuals.

Methodology and Principal Findings

Here, we show that nelfinavir generates oxidative stress in the parasite, leading to altered physiological parameters such as an increase in the sub-G1 DNA content, nuclear DNA fragmentation and loss of mitochondrial potential, which are all characteristics of apoptosis. Pretreatment of axenic amastigotes with the caspase inhibitor z-VAD-fmk did not inhibit the increase in sub-G1 DNA content in nelfinavir-treated parasites, suggesting therefore that this antiviral agent does not kill Leishmania amastigotes in a caspase-dependent manner. Furthermore, we observed that the mitochondrial resident protein endonuclease G is involved. We also demonstrate that parasites overexpressing GSH1 (the rate limiting enzyme of glutathione biosynthesis) were more resistant to nelfinavir when compared to untransfected controls.

Conclusions and Significance

These data suggest that nelfinavir induces oxidative stress in Leishmania amastigotes, culminating in caspase-independent apoptosis, in which DNA is degraded by endonuclease G. This study provides a rationale for future, long-term design of new therapeutic strategies to test nelfinavir as a potential antileishmanial agent as well as for possible future use in Leishmania/HIV-1 coinfections.     

The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression

Background

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.

Methodology/Principal Findings

Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.

Conclusions/Significance

Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.     

Identification of Pathway-Biased and Deleterious Melatonin Receptor Mutants in Autism Spectrum Disorders and in the General Population

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration of the melatonin pathway has been reported in circadian disorders, diabetes and autism spectrum disorders (ASD). However, very little is known about the genetic variability of melatonin receptors in humans. Here, we sequenced the melatonin receptor MTNR1A and MTNR1B, genes coding for MT₁ and MT₂ receptors, respectively, in a large panel of 941 individuals including 295 patients with ASD, 362 controls and 284 individuals from different ethnic backgrounds. We also sequenced GPR50, coding for the orphan melatonin-related receptor GPR50 in patients and controls. We identified six non-synonymous mutations for MTNR1A and ten for MTNR1B. The majority of these variations altered receptor function. Particularly interesting mutants are MT₁-I49N, which is devoid of any melatonin binding and cell surface expression, and MT₁-G166E and MT₁-I212T, which showed severely impaired cell surface expression. Of note, several mutants possessed pathway-selective signaling properties, some preferentially inhibiting the adenylyl cyclase pathway, others preferentially activating the MAPK pathway. The prevalence of these deleterious mutations in cases and controls indicates that they do not represent major risk factor for ASD (MTNR1A case 3.6% vs controls 4.4%; MTNR1B case 4.7% vs 3% controls). Concerning GPR50, we detected a significant association between ASD and two variations, Δ502–505 and T532A, in affected males, but it did not hold up after Bonferonni correction for multiple testing. Our results represent the first functional ascertainment of melatonin receptors in humans and constitute a basis for future structure-function studies and for interpreting genetic data on the melatonin pathway in patients.     

Combined Mutation And Rearrangement Screening by Quantitative PCR High-Resolution Melting: Is It Relevant for Hereditary Recurrent Fever Genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.